Research Organizations

Research Summary

My research focuses on understanding the role of individual sodium channels, a class of proteins that conduct electrical currents, in the transmission of nerve impulses. A few sodium channels have been implicated in the hyperexcitability of pain-sensing neurons in acquired and inherited pain disorders. We are currently investigating the contribution of individual channels to the excitability of neurons under normal and pathological conditions. These studies aim to identify new targets for treatment of neurological disorders including neuropathic pain.

Extensive Research Description

My research has focused on
studying voltage-gated sodium channels regulation by accessory proteins and
phosphorylation, and the contribution of specific channels to electrogenesis in
dorsal root ganglion (DRG) neurons under normal conditions and in inherited channelopathies. Sodium channels are
heterotrimers consisting of a large pore-forming alpha-subunit
(referred to as channel), and smaller auxiliary beta-subunits. Sodium channels are large polypeptides
(1700-2000 amino acids) which fold into four domains (DI-DIV), each domain
including six transmembrane segments, linked by three loops (L1-L3). Nine alpha-subunits
(Nav1.1-Nav1.9) encoded by the SCN1A-SCN5A and SCN8A-SCN11A
genes, have been identified in mammals, and their expression is spatially-, and
temporally-regulated. Different channels
activate and inactivate with different kinetics and voltage-dependent
properties, with six channels (Nav1.1-1.4, Nav1.6 and Nav1.7) sensitive to
block by nanomolar concentrations of tetrodotoxin (TTX-S), and three channels (Nav1.5,
Nav1.8 and Nav1.9) resistant to this blocker (TTX-R). Because channel properties are cell-type
dependent and sodium channel properties can be modulated in a
cell-type-specific manner, we have developed methods to study these channels within
native neurons.

Neuronal sodium channels, Nav1.3,
Nav1.6, Nav1.7, Nav1.8 and Nav1.9
have been intensively investigated because of their potential role in nervous
system disorders. Specifically,
Nav1.6-Nav1.9 are the main channels in DRG neurons, and their altered
expression and modulation following injury or inflammation have been linked to
acquired neuropathic pain in animal models.
Electrophysiological studies over the past decade from our group and
several other research groups have attributed specific roles for individual
channels to specific aspects of action potential firing. Thus, in small-diameter nociceptive neurons Nav1.7
and Nav1.9 are considered threshold channels that boost weak stimuli and Nav1.8
is the channel that carries the main sodium current of action potentials with
Nav1.6 contributing to the first few spikes.
In large-diameter DRG neurons Nav1.6 is the main sodium channel with
Nav1.7 and Nav1.8 present in a smaller number of these cells. Recently, mutations in Nav1.7 have been shown
to underlie two distinct pain disorders, while its complete loss results in
congenital insensitivity to pain. Ongoing
studies aim to better understand the contribution of these channels to the
pathophysiology of pain and other neurological disorders, including multiple
sclerosis and spinal cord injury.

Using genetic, biochemical, and
electrophysiological approaches we have identified channel partners that may be
important for channel trafficking and /or modulation. We have shown that members of the intracellular
fibroblast growth factors (FGF11-14) can regulate biophysical properties of sodium
channels Nav1.2, Nav1.3, Nav1.6 and Nav1.7.
We have also identified and characterized CAP-1A, a cytosolic protein
which binds selectively to Nav1.8, among sodium channels, and induce
a reduction in the current density in DRG neurons. CAP-1A also binds to clathrin, and may
represent a new class of adaptor proteins which link sodium channels and
clathrin and regulate sodium channel density by clathrin-mediated
endocytosis. We have also reported that
contactin, a GPI-anchored cell adhesion moleculae, regulates the sodium channel
density of Nav1.3, Nav1.8 and Nav1.9, but not
Nav1.6 and Nav1.7.
We are continuing this line of investigation to investigate
isoform-specific regulation of sodium channels by these, and other
newly-discovered channel partners.

Phosphorylation
of ion channels is a rapid and reversible that may significantly alter neuronal
physiology, and phosphorylation of sodium channels is predicted to acutely regulate
DRG neuron firing under pathological conditions. It is well-established that tissue and nerve
injury cause the release of pro-nociceptive cytokines and growth factors, and
alter ion conductances, leading to sensitization and hyperexcitability of
nociceptive neurons. For example, TNF-a, a major pro-nociceptive cytokine and other
pro-nociceptive factors including neurotrophic growth factor (NGF) activate
downstream signaling pathways including the mitogen-activated protein kinase
(MAPK) p38 (stress activated MAPK) and ERK1/2 (extracellular regulated kinase),
a process which has been implicated in inducing hyperexcitability of injured
DRG neurons. In fact, published work has
shown that acute application of TNF-a
to DRG neurons in culture increases the TTX-R current density in a
p38-dependent manner. These results
suggest that Nav1.8 current density may be regulated by activated
p38. We have designed experiments to answer the question of whether the Nav1.8 channel is
a substrate for direct phosphorylation by activated p38 MAPK, or whether
phosphorylation of the channel is necessary for the increase in current
density. Additional studies aim to investigate the effect of p38 and ERK1/2 on different sodium channel
isoforms that are co-expressed within the same neuron.

MAP
kinases are proline-directed serine/threonine kinases which phosphorylate SP or
TP sites in their substrates. We have
identified several potential MAPK phosphoacceptor sites within cytoplasmic
regions of sodium channels, suggesting that they may be MAPK substrates during
pain signal transduction. Using in vitro
kinase assays on individual channel fragments, we have now shown that loop 1
(L1), which joins domains I and II, carries a single p38 phosphorylation site in
Nav1.6 and two sites in Nav1.8.
Interestingly these phosphoacceptor sites are part of a PXSP motif, the
minimal PXXP motif that binds proteins with SH3 domains. Also PXpSP motif is a potential binding motif
of the type 4 WW domain of some ubiquitin ligases. Thus phosphorylation of these sites within
Nav1.6 and Nav1.8 may act as a switch that permits binding or un-binding of
channel partners, leading to regulation of these sodium channels. Indeed, we have shown that activation of p38
increases Nav1.8 current density while it reduces Nav1.6 current
density. Our findings suggest that p38
directly modulates Nav1.6 and Nav1.8 in vivo, providing a rapid mechanism that can regulate nociceptive
neuron excitability following injury.
Ongoing studies aim to elucidate mechanisms that underlie the
p38-mediated, isoform-specific regulation of sodium channels, and to
investigate the effect of p38 and ERK1/2 on other sodium channels within DRG
neurons.

While the role of sodium channels
in acquired channelopathies leading to neuropathic pain is well-established,
their role in inherited painful neuropathies has been less clear. However, the recent discovery of a monogenic
link of Nav1.7 to pain disorders in humans provided a compelling
case for establishing Nav1.7 as central to pain-signaling. Dominant gain-of-function mutations in SCN9A, the gene that encodes sodium
channel Nav1.7, have been linked to two severe pain syndromes,
inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD),
while recessive loss-of-function mutations have been linked to complete
insensitivity to pain (CIP).
Electrophysiological characterization of these mutations has elucidated molecular
basis for altered excitability of DRG neurons that express these mutant channels,
thus establishing a mechanistic link to human pain.

Pain in IEM is triggered by warmth
and is localized to the distal extremities (feet and hands). IEM has been reported as early as 1-year-old
(early-onset), in the second decade (delayed-onset), and in adults
(adult-onset). Although mutations in Nav1.7
have been identified in patients with early- and delayed-onset IEM, the
molecular basis of adult-onset IEM remains elusive. Our studies have shown that all IEM mutations in Nav1.7 characterized
thus far shift activation voltage-dependence in a hyperpolarized direction,
allowing the mutant channels to open (activate) in response to a weaker
stimulus, compared to wild-type channels.
At the cellular level, mutant Nav1.7 channels studied thus
far lower threshold for single action potentials and increase firing frequency
in DRG neurons, with all but one causing a depolarizing shift in resting
potential. While pharmacotherapy for IEM
is largely ineffective, cooling is effective in relieving pain although
cold-induced injury is a serious outcome of this practice. A recent study from our group, however, identified
a new IEM case in which patients responded favorably to the anticonvulsant drug
carbamazepine. Surprisingly, while carbamazepine
blocked mutant and wild-type channels equally, it induced a depolarizing shift
of activation of the mutant channel, suggesting an allosteric effect of the
drug on the mutant channel.

A second set of mutations of Nav1.7
underlies many of the PEPD cases. Severe
perirectal pain in PEPD along with skin flushing can start in infancy and
possibly in utero, but with no
reported involvement of feet and hands. While
seizures and cardiac symptoms may accompany PEPD, a link to the mutant Nav1.7
channel in sympathetic neurons, which normally express this channel, has not yet
been established. As patients age, pain
extends to ocular and maxillary/mandibular areas and is triggered by cold,
eating or emotional state. Our group and
others have shown that the PEPD mutations shift the voltage-dependence of
steady-state fast-inactivation of Nav1.7 in a depolarizing direction and, depending
upon the specific mutation, may make inactivation incomplete resulting in a
persistent current. PEPD symptoms, in
contrast to IEM, are well controlled by carbamazepine; impaired inactivation of
PEPD Nav1.7 mutant channels could explain the favorable response of
the patients to carbamazepine. At the
cellular level, we have shown that PEPD Nav1.7 mutant channels lower
threshold for single action potential and increase frequency of firing in DRG
neurons, but without altering resting potential.

Together with the published data that
have shown that Nav1.7-related CIP is not accompanied by other major
sensory deficits, except impaired olfaction, motor or cognitive deficits, our data
provide a compelling rationale to target Nav1.7 for the development of new pain
therapeutic agents which are predicted to have minimal side effects.

Identification and characterization of mutations in peripheral voltage-gated sodium channels in patients with heritable pain disorders.

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